Neutralizing antibodies against GDF-8 and uses therefor

ABSTRACT

The disclosure provides novel antibodies against growth and differentiation factor-8 (GDF-8), in particular human antibodies, and antibody fragments, including those that inhibit GDF-8 activity in vitro and/or in vivo. The disclosure also provides methods for diagnosing, preventing, or treating degenerative disorders of muscle or bone, or disorders of insulin metabolism.

This application claims priority to U.S. provisional Ser. No.60/419,964, filed Oct. 22, 2002, which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The technical field relates to antibodies against growth anddifferentiation factor-8 (GDF-8), in particular human antibodies, andantibody fragments, especially those that inhibit GDF-8 activity invitro and/or in vivo. The field further relates to diagnosing,preventing, or treating degenerative disorders of muscle or bone, ordisorders of insulin metabolism.

BACKGROUND

Growth and differentiation factor-8 (GDF-8), also known as myostatin, isa secreted protein and is a member of the transforming growthfactor-beta (TGF-β) superfamily of structurally related growth factors,all of which possess physiologically important growth-regulatory andmorphogenetic properties (Kingsley et al. (1994) Genes Dev., 8: 133-146;Hoodless et al. (1998) Curr. Topics Microbiol. Immunol., 228: 235-272).Similarly to TGF-β, human GDF-8 is synthesized as a 375 amino acid longprecursor protein. The precursor GDF-8 protein forms a homodimer. Duringprocessing the amino-terminal propeptide is cleaved off at Arg-266. Thecleaved propeptide, known as the “latency-associated peptide” (LAP), mayremain noncovalently bound to the homodimer, thereby inactivating thecomplex (Miyazono et al. (1988) J. Biol. Chem., 263: 6407-6415;Wakefield et al. (1988) J. Biol. Chem., 263: 7646-7654; Brown et al.(1990) Growth Factors, 3: 35-43; and Thies et al. (2001) Growth Factors,18: 251-259). The complex of mature GDF-8 with propeptide is commonlyreferred to as the “small latent complex” (Gentry et al. (1990)Biochemistry, 29: 6851-6857; Derynck et al. (1995) Nature, 316: 701-705;and Massague (1990) Ann. Rev. Cell Biol., 12: 597-641). Other proteinsare also known to bind to mature GDF-8 and inhibit its biologicalactivity. Such inhibitory proteins include follistatin andfollistatin-related proteins (Gamer et al. (1999) Dev. Biol., 208:222-232).

An alignment of deduced amino acid sequences from various speciesdemonstrates that GDF-8 is highly conserved throughout evolution(McPherron et al. (1997) Proc. Nat. Acad. Sci. U.S.A., 94: 12457-12461).In fact, the sequences of human, mouse, rat, porcine, and chicken GDF-8are 100% identical in the C-terminal region, while in baboon, bovine,and ovine they differ only by 3 amino acids. The zebrafish GDF-8 is themost diverged; however, it is still 88% identical to human.

The high degree of conservation suggests that GDF-8 has an essentialfunction. GDF-8 is highly expressed in the developing and adult skeletalmuscle and was found to be involved in the regulation of criticalbiological processes in the muscle and in osteogenesis. For example,GDF-8 knockout transgenic mice are characterized by a marked hypertrophyand hyperplasia of the skeletal muscle (McPherron et al. (1997) Nature,387: 83-90) and altered cortical bone structure (Hamrick et al. (2000)Bone, 27 (3): 343-349). Similar increases in skeletal muscle mass areevident in naturally occurring mutations of GDF-8 in cattle (Ashmore etal. (1974) Growth, 38: 501-507; Swatland et al. (1994) J. Anim. Sci.,38: 752-757; McPherron et al. (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461; and Kambadur et al. (1997) Genome Res., 7: 910-915).Studies have indicated that muscle wasting associated with HIV-infectionis accompanied by an increase in GDF-8 expression (Gonzalez-Cadavid etal. (1998) Proc. Nat. Acad. Sci. U.S.A., 95: 14938-14943). GDF-8 hasalso been implicated in the production of muscle-specific enzymes (e.g.,creatine kinase) and proliferation of myoblast cells (WO 00/43781). Inaddition to its growth-regulatory and morphogenetic properties, GDF-8 isthought to be also involved in a number of other physiologicalprocesses, including glucose homeostasis in the development of type 2diabetes, impaired glucose tolerance, metabolic syndromes (e.g.,syndrome X), insulin resistance induced by trauma, such as burns ornitrogen imbalance, and adipose tissue disorders (e.g., obesity) (Kim etal. (2001) BBRC, 281: 902-906).

A number of human and animal disorders are associated with functionallyimpaired muscle tissue, e.g., muscular dystrophy (including Duchenne'smuscular dystrophy), amyotrophic lateral sclerosis (ALS), muscleatrophy, organ atrophy, frailty, congestive obstructive pulmonarydisease, sarcopenia, cachexia, and muscle wasting syndromes caused byother diseases and conditions. To date, very few reliable or effectivetherapies have been developed to treat these disorders.

There are also a number of conditions associated with a loss of bone,which include osteoporosis and osteoarthritis, especially in the elderlyand/or postmenopausal women. In addition, metabolic bone diseases anddisorders include low bone mass due to chronic glucocorticoid therapy,premature gonadal failure, androgen suppression, vitamin D deficiency,secondary hyperparathyroidism, nutritional deficiencies, and anorexianervosa. Currently available therapies for these conditions work byinhibiting bone resorption. A therapy that promotes new bone formationwould be a desirable alternative to these therapies.

Thus, a need exists to develop new therapies that contribute to anoverall increase of muscle mass and/or strength and/or bone density,especially, in humans.

SUMMARY

It is one of the objects of the present invention to provide safe andeffective therapeutic methods for muscle and/or bone-associateddisorders.

It is another object of the invention to provide methods of increasingmuscle mass and/or bone strength and/or density in vertebrates.

It is yet another object of the invention to provide inhibitors of GDF-8that are safe and effective in vivo.

Still another object of the invention is to provide human antibodies andfragments thereof that bind GDF-8 with high specificity and affinity.

Thus, methods for treating muscle and bone degenerative disorders areprovided. The methods are also useful for increasing muscle mass andbone density in normal animals. Also provided are novel human anti-GDF-8antibodies, termed Myo29, Myo28, and Myo22, and antibodies andantigen-binding fragments derived therefrom. The antibodies of theinvention possess a number of useful properties. First, the antibodiesare capable of binding mature GDF-8 with high affinity. Second, thedisclosed antibodies inhibit GDF-8 activity in vitro and in vivo asdemonstrated, for example, by inhibition of ActRIIB binding and reportergene assays. Third, the disclosed antibodies may inhibit GDF-8 activityassociated with negative regulation of skeletal muscle mass and bonedensity.

Certain embodiments of the invention comprise the V_(H) and/or V_(L)domain of the Fv fragment of Myo29, Myo28, or Myo22. Further embodimentscomprise one or more complementarity determining regions (CDRs) of anyof these V_(H) and V_(L) domains. Other embodiments comprise an H3fragment of the V_(H) domain of Myo29, Myo28, or Myo22.

Other aspects provide compositions containing antibodies of theinvention or their antigen-binding fragments, and their use in methodsof inhibiting or neutralizing GDF-8, including methods of treatment ofthe human or animals. The antibodies of the invention may be used totreat or prevent conditions in which an increase in muscle tissue orbone density is desirable. For example, the presently disclosedantibodies may be used in therapies to repair damaged muscle, e.g.,myocardium, diaphragm, etc. Exemplary disease and disorders includemuscle and neuromuscular disorders such as muscular dystrophy (includingDuchenne's muscular dystrophy); amyotrophic lateral sclerosis; muscleatrophy; organ atrophy; frailty; tunnel syndrome; congestive obstructivepulmonary disease; sarcopenia, cachexia, and other muscle wastingsyndromes; adipose tissue disorders (e.g., obesity); type 2 diabetes;impaired glucose tolerance; metabolic syndromes (e.g., syndrome X);insulin resistance induced by trauma such as burns or nitrogenimbalance; and bone degenerative diseases (e.g., osteoarthritis andosteoporosis).

In addition, the presently disclosed antibodies may be used as adiagnostic tool to quantitatively or qualitatively detect GDF-8 or itsfragments in a biological sample. The presence or amount of GDF-8detected can be correlated with one or more of the medical conditionslisted above.

Another aspect provides an isolated nucleic acid, which comprises asequence encoding a V_(H) or V_(L) domain from an Fv fragment of Myo29,Myo28, or Myo22. An isolated nucleic acid, which comprises a sequenceencoding at least one CDR from any of the presently disclosed V_(H) andV_(L) domains, is also disclosed. Another aspect provides host cellscomprising such nucleic acid.

Yet another aspect provides a method of producing new V_(H) and V_(L)domains and/or functional antibodies comprising all or a portion of suchdomains derived from the V_(H) or V_(L) domains of Myo29, Myo28, orMyo22.

Additional objects of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Variousobjects, aspects, and advantages of the invention will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that biotinylated GDF-8 and BMP-11 bind the ActRIIBreceptor with an ED₅₀ of 15 ng/ml and 40 ng/ml, respectively.

FIG. 2 shows inhibition of GDF-8 binding to the ActRIIB receptor by scFvfragments of the invention. As illustrated, the IC₅₀ for scFv's ofMyo29, Myo28, and Myo22 are 2.4 nM, 1.7 nM, and 60 nM, respectively.

FIGS. 3A and 3B show that preincubation of Myo29 with biotinylated GDF-8or BMP-11 at 10 ng/ml inhibits GDF-8 or BMP-11 binding to ActRIIB in theActRIIB binding assay with an IC₅₀ of 0.2-0.4 nM.

FIGS. 4B and 4C depict results of pGL3(CAGA)₁₂ reporter gene assays, inwhich Myo29 was tested. FIG. 4A demonstrates the baseline conditions,i.e., induction of the reporter gene activity by GDF-8, BMP-11, andactivin. FIGS. 4B and 4C show that Myo29 reduces the GDF-8 activity in adose-responsive manner, with an IC₅₀ of 15-30 ng/ml, and inhibits thebiological activity of BMP-11 to the same extent. FIG. 4D illustratesthat Myo29 does not affect the activity of activin in this assay.

FIG. 5 shows results of epitope mapping for Myo22, Myo28, and Myo29. Theepitope for Myo29 was mapped from amino acid 72 to amino acid 88 ofmature GDF-8; for Myo22, from amino acid 1 to amino acid 44; for Myo28,from amino acids 1 to amino acid 98.

FIG. 6 demonstrates results of a substitution analysis of the Myo29epitope. Residues Lys-78, Pro-81, and Asn-83 in mature GDF-8 appear tobe important for Myo29 binding to GDF-8.

FIG. 7 depicts results of an immunoprecipitation experiment performedwith Myo29 and Myo28. Conditioned medium from CHO cells expressingGDF-8, which were radiolabeled with ³⁵S-methionine/cysteine, wassubjected to immunoprecipitation with Myo29 or Myo28. Theimmunoprecipitates were then analyzed by SDS-PAGE under reducingconditions. Bands on the gel are identified as mature GDF-8, GDF-8propeptide, and unprocessed GDF-8.

FIG. 8 depicts results of a pharmacokinetic study in which C57B6/SCIDmice received a dose of 1 mg/kg as a single intravenous (IV) orintraperitoneal (IP) administration of Myo29. Myo29 shows prolongedterminal half-life of around one week and low clearance around 1ml/hr/kg. The fraction absorbed following IP injection is about 77%.

FIG. 9 shows comparisons of quadriceps mass in male C57B6/SCID micetreated weekly with various doses of Myo29 (60, 10, and 1 mg/kg), orvehicle (PBS). Treatment with Myo29, at the 10 and 60 mg/kg dose levelsfor four weeks results in a statistically significant increase in musclemass of 19% and 23%, respectively.

FIGS. 10A and 10B show gastrocnemius and quadriceps mass in female CB17SCID mice treated weekly with various doses of Myo29 (10, 5, 2.5, and 1mg/kg) or PBS for four weeks. Muscle mass is increased by 10 to 20% inmice treated with Myo29 as compared to the vehicle control.

FIGS. 11A and 11B demonstrate respectively gastrocnemius and quadricepsmuscle mass in female CB17 SCID mice treated weekly with various dosesof Myo29 (10, 5, 2.5, and 1 mg/kg) or PBS for twelve weeks. Mice treatedwith Myo29 show increases in muscle mass ranging from 12 to 28%.

FIG. 12 shows the front limb muscle strength, as measured by a gripstrength meter, in female CB17 SCID mice treated weekly with Myo29 (10and 5 mg/kg) or PBS for twelve weeks. Front limb strength is increasedby 17% and 23% in mice treated with Myo29 at 5 mg/kg and 10 mg/kg,respectively.

DETAILED DESCRIPTION

I. Definitions

The term “antibody,” as used herein, refers to an immunoglobulin or apart thereof, and encompasses any polypeptide comprising anantigen-binding site regardless of the source, species of origin, methodof production, and characteristics. As a non-limiting example, the term“antibody” includes human, orangutan, mouse, rat, goat, sheep, andchicken antibodies. The term includes but is not limited to polyclonal,monoclonal, monospecific, polyspecific, non-specific, humanized,single-chain, chimeric, synthetic, recombinant, hybrid, mutated, andCDR-grafted antibodies. For the purposes of the present invention, italso includes, unless otherwise stated, antibody fragments such as Fab,F(ab′)₂, Fv, scFv, Fd, dAb, and other antibody fragments that retain theantigen-binding function.

Antibodies can be made, for example, via traditional hybridomatechniques (Kohler and Milstein (1975) Nature, 256: 495-499),recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage displaytechniques using antibody libraries (Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597). For variousother antibody production techniques, see Antibodies: A LaboratoryManual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988.

The term “antigen-binding domain” refers to the part of an antibodymolecule that comprises the area specifically binding to orcomplementary to a part or all of an antigen. Where an antigen is large,an antibody may only bind to a particular part of the antigen. The“epitope” or “antigenic determinant” is a portion of an antigen moleculethat is responsible for specific interactions with the antigen-bindingdomain of an antibody. An antigen-binding domain may be provided by oneor more antibody variable domains (e.g., a so-called Fd antibodyfragment consisting of a V_(H) domain). An antigen-binding domaincomprises an antibody light chain variable region (V_(L)) and anantibody heavy chain variable region (V_(H)).

The term “repertoire” refers to a genetically diverse collection ofnucleotides, e.g., DNA, sequences derived wholly or partially fromsequences which encode expressed immunoglobulins. The sequences aregenerated by in vivo rearrangement of, e.g., V, D, and J segments for Hchains and, e.g., V and J segment for L chains. Alternatively, thesequences may be generated from a cell line by in vitro stimulation andin response to which rearrangement occurs. Alternatively, part or all ofthe sequences may be obtained by combining, e.g., unrearranged Vsegments with D and J segments, by nucleotide synthesis, randomisedmutagenesis, and other methods as disclosed in U.S. Pat. No. 5,565,332.

The term “specific interaction,” or “specifically binds,” or the like,means that two molecules form a complex that is relatively stable underphysiologic conditions. The term is also applicable where, e.g., anantigen-binding domain is specific for a particular epitope, which iscarried by a number of antigens, in which case the antibody carrying theantigen-binding domain will be able to bind to the various antigenscarrying the epitope. Thus, an antibody may specifically bind, forexample, BMP-11 and GDF-8 as long as it binds to the epitope, which iscarried by both.

Specific binding is characterized by a high affinity and a low tomoderate capacity. Nonspecific binding usually has a low affinity with amoderate to high capacity. Typically, the binding is considered specificwhen the affinity constant K_(a) is higher than 10⁶ M⁻¹, or preferablyhigher than 10⁸ M⁻¹. If necessary, non-specific binding can be reducedwithout substantially affecting specific binding by varying the bindingconditions. Such conditions are known in the art, and a skilled artisanusing routine techniques can select appropriate conditions. Theconditions are usually defined in terms of concentration of antibodies,ionic strength of the solution, temperature, time allowed for binding,concentration of non-related molecules (e.g., serum albumin, milkcasein), etc. Exemplary conditions are set forth in Examples 4, 7, and10.

The phrase “substantially as set out” means that the relevant CDR,V_(H), or V_(L) domain will be either identical or highly similar to thespecified regions of which the sequence is set out herein. For example,such substitutions include 1 or 2 out of any 5 amino acids in thesequence of a CDR (H1, H2, H3, L1, L2, or L3).

The term “TGF-β superfamily” refers to a family of structurally-relatedgrowth factors. This family of related growth factors is well known inthe art (Kingsley et al. (1994) Genes Dev., 8: 133-146; Hoodless et al.(1998) Curr. Topics Microbiol. Immunol., 228: 235-72). The TGF-βsuperfamily includes bone morphogenetic proteins (BMP), activin,inhibin, mullerian inhibiting substance, glial-derived neurotrophicfactor, and a still growing number of growth and differentiation factors(GDF), such as GDF-8 (myostatin). Many of such proteins are structurallyand/or functionally related to GDF-8. For example, human BMP-11, alsoknown as GDF-11, is 90% identical to GDF-8 at the amino-acid level(Gamer et al. (1999) Dev. Biol. 208, 222-232; Nakshima et al. (1999)Mech. Dev., 80: 185-189).

The term “GDF-8” refers to a specific growth and differentiationfactor-8 and, where appropriate, factors that are structurally orfunctionally related to GDF-8, for example, BMP-11 and other factorsbelonging to the TGF-β superfamily. The term refers to the full-lengthunprocessed precursor form of GDF-8 as well as the mature and propeptideforms resulting from post-translational cleavage. The term also refersto any fragments and variants of GDF-8 that maintain at least somebiological activities associated with mature GDF-8, as discussed herein,including sequences that have been modified. The amino acid sequence ofmature human GDF-8 is provided in SEQ ID NO:49. The present inventionrelates to GDF-8 from all vertebrate species, including, but not limitedto, human, bovine, chicken, mouse, rat, porcine, ovine, turkey, baboon,and fish (for sequence information, see, e.g., McPherron et al. (1997)Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461).

The term “mature GDF-8” refers to the protein that is cleaved from thecarboxy-terminal domain of the GDF-8 precursor protein. The mature GDF-8may be present as a monomer, homodimer, or in a GDF-8 latent complex.Depending on conditions, mature GDF-8 may establish equilibrium betweenany or all of these different forms. In its biologically active form,the mature GDF-8 is also referred to as “active GDF-8.”

The term “GDF-8 propeptide” refers to the polypeptide that is cleavedfrom the amino-terminal domain of the GDF-8 precursor protein. The GDF-8propeptide is capable of binding to the propeptide binding domain on themature GDF-8.

The term “GDF-8 latent complex” refers to the complex of proteins formedbetween the mature GDF-8 homodimer and the GDF-8 propeptide. It isbelieved that two GDF-8 propeptides associate with two molecules ofmature GDF-8 in the homodimer to form an inactive tetrameric complex.The latent complex may include other GDF inhibitors in place of or inaddition to one or more of the GDF-8 propeptides.

The term “GDF-8 activity” refers to one or more of physiologicallygrowth-regulatory or morphogenetic activities associated with activeGDF-8 protein. For example, active GDF-8 is a negative regulator ofskeletal muscle mass. Active GDF-8 can also modulate the production ofmuscle-specific enzymes (e.g., creatine kinase), stimulate myoblastproliferation, and modulate preadipocyte differentiation to adipocytes.Exemplary procedures for measuring GDF-8 activity in vivo and in vitroare set forth in Examples 2, 3, 6, and 13.

The term “GDF-8 inhibitor” includes any agent capable of inhibitingactivity, expression, processing, or secretion of GDF-8. Such inhibitorsinclude proteins, antibodies, peptides, peptidomimetics, ribozymes,anti-sense oligonucleotides, double-stranded RNA, and other smallmolecules, which specifically inhibit GDF-8. Such inhibitors are said to“inhibit,” “neutralize,” or “reduce” the biological activity of GDF-8.

The terms “neutralize,” “neutralizing,” “inhibitory,” and their cognatesrefer to a reduction in the activity of GDF-8 by a GDF-8 inhibitor,relative to the activity of GDF-8 in the absence of the same inhibitor.The reduction in activity is preferably at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or higher.

The term “treatment” is used interchangeably herein with the term“therapeutic method” and refers to both therapeutic treatment andprophylactic/preventative measures. Those in need of treatment mayinclude individuals already having a particular medical disorder as wellas those who may ultimately acquire the disorder (i.e., those needingpreventative measures).

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term refers topreparations where the isolated protein is sufficiently pure to beadministered as a therapeutic composition, or at least 70% to 80% (w/w)pure, more preferably, at least 80%-90% (w/w) pure, even morepreferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%,98%, 99%, or 100% (w/w) pure.

The term “mammal” refers to any animal classified as such, includinghumans, domestic and farm animals, zoo, sports, or pet animals, such asdogs, horses, cats, sheep, pigs, cows, etc.

The term “effective dose,” or “effective amount,” refers to that amountof the compound that results in amelioration of symptoms in a patient ora desired biological outcome (e.g., increasing skeletal muscle massand/or bone density). Such amount should be sufficient to reduce theactivity of GDF-8 associated with negative regulation of skeletal musclemass and bone density. The effective amount can be determined asdescribed in the subsequent sections.

II. Antibodies Against GDF-8 and Antigen-Binding Fragments

A. Human Antibodies Myo29, Myo28, and Myo22

The present disclosure provides novel antibodies against GDF-8, andantigen-binding fragments thereof. Nonlimiting illustrative embodimentsof such antibodies are termed Myo29, Myo28, and Myo22. These exemplaryembodiments are provided in the form of human IgG₁ antibodies.

The antibodies of the invention possess unique and beneficialcharacteristics. First, these antibodies are capable of binding matureGDF-8 with high affinity. Second, the antibodies of the invention mayinhibit GDF-8 activity in vitro and in vivo as demonstrated, forexample, by inhibition of ActRIIB binding and reporter gene assays. Theantibodies of the present invention are also capable of specificallybinding and/or inhibiting activity of BMP-11 as demonstrated, forexample, by inhibition of ActRIIB binding and reporter gene assays.Third, the disclosed antibodies may inhibit GDF-8 activity associatedwith negative regulation of skeletal muscle mass and bone density.

In an exemplary embodiment, the presently disclosed antibodies arecapable of specifically binding to both GDF-8 and BMP-11. It iscontemplated that the antibodies may also react with other proteins, forexample, those belonging to the TGF-β superfamily such as mullerianinhibiting substance, glial-derived neurotrophic factor, or growth anddifferentiation factors other than GDF-8. In certain embodiments, Myo29reacts with a protein comprising a sequence identical to amino acid 72to 88 of SEQ ID NO:49. In further embodiments, Myo29 binds to a proteincomprising the sequence Lys-Xaa1-Xaa2-Pro-Xaa3-Asn (SEQ ID NO:54),wherein Xaa1, Xaa2, and Xaa3 each is any amino acid. In furtherembodiments, at least one of the following conditions is met: (1)Xaa1=Met, (2) Xaa2=Ser, and (3) Xaa3=lie; all independently of eachother. In other embodiments, Myo22 recognizes an epitope within thefirst 44 N-terminal amino acids in the sequence of mature GDF-8 (aminoacids 1 through 44 of SEQ ID NO:49).

One of ordinary skill in the art will recognize that antibodies of theinvention may be used to detect, measure, and inhibit proteins thatdiffer from those stated above. In general, antibodies of the inventioncan be used with any protein that comprises a sequence which is at leastabout 70%, 80%, 90%, 95%, or more identical to any sequence of at least100, 80, 60, 40, or 20 of contiguous amino acids in the sequence of themature form of GDF-8 set forth SEQ ID NO:49. Nonlimiting examples ofsuch proteins include sequences of GDF-8 derived from various species,which are described in the present specification. The percent identityis determined by standard alignment algorithms such as, for example,Basic Local Alignment Tool (BLAST) described in Altschul et al. (1990)J. Mol. Biol., 215: 403-410, the algorithm of Needleman et al. (1970) J.Mol. Biol., 48: 444-453, or the algorithm of Meyers et al. (1988)Comput. Appl. Biosci., 4: 11-17.

B. Variable Domains

Intact antibodies, also known as immunoglobulins, are typicallytetrameric glycosylated proteins composed of two light (L) chains ofapproximately 25 kDa each and two heavy (H) chains of approximately 50kDa each. Two types of light chain, termed λ and κ, are found inantibodies. Depending on the amino acid sequence of the constant domainof heavy chains, immunoglobulins can be assigned to five major classes:A, D, E, G, and M, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known in the art. For a review ofthe antibody structure, see Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, eds. Harlow et al., 1988. Briefly, each light chainis composed of an N-terminal variable (V) domain (V_(L)) and a constant(C) domain (C_(L)). Each heavy chain is composed of an N-terminal Vdomain, three or four C domains, and a hinge region. The C_(H) domainmost proximal to V_(H) is designated as C_(H)1. The V_(H) and V_(L)domain consist of four regions of relatively conserved sequence calledframework regions (FR1, FR2, FR3, and FR4), which form a scaffold forthree regions of hypervariable sequence (complementarity determiningregions, CDRs). The CDRs contain most of the residues responsible forspecific interactions with the antigen. CDRs are referred to as CDR1,CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain arereferred to as H1, H2, and H3, while CDR constituents on the light chainare referred to as L1, L2, and L3. CDR3 is the greatest source ofmolecular diversity within the antibody-binding site. H3, for example,can be as short as two amino acid residues or greater than 26. Thesmallest antigen-binding fragment is the Fv, which consist of the V_(H)and V_(L) domains. The Fab fragment (Fragment antigen binding) consistsof the V_(H)-C_(H)1 and V_(L)-C_(L) domains covalently-linked by adisulfide bond between the constant regions. To overcome the tendency ofnon-covalently linked V_(H) and V_(L) domains in the Fv to dissociatewhen co-expressed in a host cell, a so-called single chain (sc) Fvfragment (scFv) can be constructed, in which a flexible and adequatelylong polypeptide links either the C-terminus of the V_(H) to theN-terminus of the V_(L) or the C-terminus of the V_(L) to the N-terminusof the V_(H). The most commonly used linker has been a 15-residue(Gly₄Ser)₃ peptide, but other linkers are also known in the art.

Antibody diversity is created by the use of multiple germline genesencoding variable regions and a variety of somatic events. The somaticevents include recombination of variable gene segments with diversity(D) and joining (J) gene segments to make a complete V_(H) region andthe recombination of variable and joining gene segments to make acomplete V_(L) region. The recombination process itself is imprecise,resulting in the loss or addition of amino acids at the V(D)J junctions.These mechanisms of diversity occur in the developing B cell prior toantigen exposure. After antigenic stimulation, the expressed antibodygenes in B cells undergo somatic mutation. Based on the estimated numberof germline gene segments, the random recombination of these segments,and random V_(H)-V_(L) pairing, up to 1.6×10⁷ different antibodies couldbe produced (Fundamental Immunology, 3rd ed., ed. Paul, Raven Press, NewYork, N.Y., 1993). When other processes which contribute to antibodydiversity (such as somatic mutation) are taken into account, it isthought that upwards of 1×10¹⁰ different antibodies could be generated(Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, SanDiego, Calif., 1995). Because of the many processes involved ingenerating antibody diversity, it is unlikely that independently derivedmonoclonal antibodies with the same antigen specificity will haveidentical amino acid sequences.

Thus, the present invention further provides novel CDRs derived fromhuman immunoglobulin gene libraries. The structure for carrying a CDR ofthe invention will generally be an antibody heavy or light chainsequence or a substantial portion thereof, in which the CDR is locatedat a location corresponding to the CDR of naturally occurring V_(H) andV_(L). The structures and locations of immunoglobulin variable domainsmay be determined as described in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services, eds. Kabat et al.,1991.

DNA and amino acid (AA) sequences of the presently disclosed antibodies,their scFV fragment, V_(H) and V_(L) domains, and CDRs are set forth inthe Sequences Listing and are enumerated as listed in Table 1. Forconvenience, the positions for each CDR within V_(H) and V_(L) domainsare listed in Table 2. The sequences of heavy and light chains excludingthe V_(H) and V_(L) domains are identical in Myo29, Myo28, and Myo22.

TABLE 1 DNA and Amino Acid Sequences of scFv, V_(H) andV_(L Domains, and CDRs) Myo29 Myo28 Myo22 DNA sequence SEQ ID NO:13 SEQID NO:7 SEQ ID NO:1 of scFv AA sequence SEQ ID NO:14 SEQ ID NO:8 SEQ IDNO:2 of scFv DNA sequence SEQ ID NO:15 SEQ ID NO:9 SEQ ID NO:3 of VH AAsequence SEQ ID NO:16 SEQ ID NO:10 SEQ ID NO:4 of VH DNA sequence SEQ IDNO:17 SEQ ID NO:11 SEQ ID NO:5 of VL AA sequence SEQ ID NO:18 SEQ IDNO:12 SEQ ID NO:6 of VL Germlined SEQ ID NO:25 SEQ ID NO:19 DNA seq. ofscFv Germlined SEQ ID NO:26 SEQ ID NO:20 AA seq. of scFv Germlined SEQID NO:27 SEQ ID NO:21 DNA seq. VH Germlined SEQ ID NO:28 SEQ ID NO:22 AAseq. of VH Germlined SEQ ID NO:29 SEQ ID NO:23 DNA seq. of VL GermlinedSEQ ID NO:30 SEQ ID NO:24 AA seq. of VL AA sequence SEQ ID NO:31 SEQ IDNO:37 SEQ ID NO:43 of H1 AA sequence SEQ ID NO:32 SEQ ID NO:38 SEQ IDNO:44 of H2 AA sequence SEQ ID NO:33 SEQ ID NO:39 SEQ ID NO:45 of H3 AAsequence SEQ ID NO:34 SEQ ID NO:40 SEQ ID NO:46 of L1 AA sequence SEQ IDNO:35 SEQ ID NO:41 SEQ ID NO:47 of L2 AA sequence SEQ ID NO:36 SEQ IDNO:42 SEQ ID NO:48 of L3

TABLE 2 Positions of CDRs within scFv's Myo29 Myo28 Myo22 CDR (SEQ IDNO:26) (SEQ ID NO:20) (SEQ ID NO:2) H1 31-35 31-35 31-35 H2 50-66 50-6650-66 H3  99-106  99-110  99-113 L1 157-167 160-173 163-176 L2 183-189189-195 192-198 L3 222-228 228-233 231-242

Presently disclosed antibodies may further comprise antibody constantregions or parts thereof. For example, a V_(L) domain may be attached atits C-terminal end to antibody light chain constant domains' includinghuman Cκ or Cλ chains, preferably Cλ chains. Similarly, a specificantigen-binding fragment based on a V_(H) domain may be attached at itsC-terminal end to all or part of an immunoglobulin heavy chain derivedfrom any antibody isotype, e.g., IgG, IgA, IgE, and IgM, and any of theisotype subclasses, particularly IgG₁ and IgG₄. In exemplaryembodiments, antibodies comprise C-terminal fragments heavy and lightchains of human IgG_(1λ). The DNA and amino acid sequences for theC-terminal fragment of the light λ chain are set forth in SEQ ID NO:50and SEQ ID NO:51, respectively. The DNA and amino acid sequences for theC-terminal fragment of IgG₁ heavy chain are set forth in SEQ ID NO:52and SEQ ID NO:53, respectively.

Certain embodiments of the invention comprise the V_(H) and/or V_(L)domain of the Fv fragment of Myo29, Myo28, or Myo22. Further embodimentscomprise one or more complementarity determining regions (CDRs) of anyof these V_(H) and V_(L) domains. One embodiment comprises an H3fragment of the V_(H) domain of Myo29, Myo28, or Myo22. The V_(H) andV_(L) domains of the invention, in certain embodiments, are germlined,i.e., the framework regions (FRs) of these domains are changed usingconventional molecular biology techniques to match the consensus aminoacid sequences of human germline gene products. In other embodiments,the framework sequences remain diverged from the germline.

C. Modified Antibodies and Their Fragments

A further aspect of the invention provides a method for obtaining anantibody antigen-binding domain specific for GDF-8. The skilled artisanwill appreciate that the antibodies of the invention are not limited tothe specific sequences of V_(H) and V_(L) as stated in Table 1 but alsoinclude variants of these sequences that retain antigen binding ability.Such variants may be derived from the provided sequences usingtechniques known in the art. Amino acid substitution, deletions, oradditions, can be made in either the FRs or in the CDRs. While changesin the framework regions are usually designed to improve stability andreduce immunogenicity of the antibody, changes in the CDRs are usuallydesigned to increase affinity of the antibody for its target. Suchaffinity-increasing changes are typically determined empirically byaltering the CDR region and testing the antibody. Such alterations canbe made according to the methods described in Antibody Engineering, 2nd.ed.; ed. Borrebaeck, Oxford University Press, 1995.

The method for making a V_(H) domain which is an amino acid sequencevariant of the V_(H) domain set out herein comprises a step of adding,deleting, substituting or inserting one or more amino acids in the aminoacid sequence of the presently disclosed V_(H) domain, optionallycombining the V_(H) domain thus provided with one or more V_(L) domains,and testing the V_(H) domain or V_(H)/V_(L) combination or combinationsfor specific binding to GDF-8, optionally, testing the ability of suchantigen-binding domain to neutralize GDF-8 activity. The V_(L) domainmay have an amino acid sequence which is substantially as set outherein.

An analogous method may be employed in which one or more sequencevariants of a V_(L) domain disclosed herein are combined with one ormore V_(H) domains.

A further aspect of the invention provides a method of preparing anantigen-binding fragment that specifically reacts with GDF-8. The methodcomprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        V_(H) domain which either include a CDR3 to be replaced or lack        a CDR3 encoding region;    -   (b) combining the repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a        V_(H) CDR3 (i.e., H3) such that the donor nucleic acid is        inserted into the CDR3 region in the repertoire so as to provide        a product repertoire of nucleic acids encoding a V_(H) domain;    -   (c) expressing the nucleic acids of the product repertoire;    -   (d) selecting a specific antigen-binding fragment specific for        GDF-8; and    -   (e) recovering the specific antigen-binding fragment or nucleic        acid encoding it.

Again, an analogous method may be employed in which a V_(L) CDR3 (i.e.,L3) of the invention is combined with a repertoire of nucleic acidsencoding a V_(L) domain, which either include a CDR3 to be replaced orlack a CDR3 encoding region.

A coding sequence CDR of the invention (e.g., CDR3) may be introducedinto a repertoire of variable domains lacking a CDR (e.g., CDR3), usingrecombinant DNA technology. For example, Marks et al. (Bio/Technology(1992) 10: 779-783) describe methods of producing repertoires ofantibody variable domains in which consensus primers directed at oradjacent to the 5′ end of the variable domain area are used inconjunction with consensus primers to the third framework region ofhuman V_(H) genes to provide a repertoire of V_(H) variable domainslacking a CDR3. The repertoire may be combined with a CDR3 of aparticular antibody. Using analogous techniques, the CDR3-derivedsequences of the present invention may be shuffled with repertoires ofV_(H) or V_(L) domains lacking a CDR3, and the shuffled complete V_(H)or V_(L) domains combined with a cognate V_(L) or V_(H) domain toprovide specific antigen-binding fragments of the invention. Therepertoire may then be displayed in a suitable host system such as thephage display system of WO 92/01047 so that suitable antigen-bindingfragments can be selected.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature (1994) 370: 389-391), who describes the technique inrelation to a β-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel V_(H) or V_(L) regionscarrying a CDR-derived sequences of the invention using randommutagenesis of one or more selected V_(H) and/or V_(L) genes to generatemutations within the entire variable domain. Such a technique isdescribed by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89:3576-3580), who used error-prone PCR.

Another method that may be used is to direct mutagenesis to CDR regionsof V_(H) or V_(L) genes. Such techniques are disclosed by Barbas et al.(Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al.(J. Mol. Biol. (1996) 263: 551-567).

Similarly, one or more, or all three CDRs may be grafted into arepertoire of V_(H) or V_(L) domains which are then screened for aspecific binding partner or binding fragments specific for GDF-8.

A substantial portion of an immunoglobulin variable domain will compriseat least the CDR regions and, optionally, their intervening frameworkregions from the scF_(v) fragments as set out herein. The portion willalso include at least about 50% of either or both of FR1 and FR4, the50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4.Additional residues at the N-terminal or C-terminal end of thesubstantial part of the variable domain may be those not normallyassociated with naturally occurring variable domain regions. Forexample, construction of specific antigen-binding fragments of thepresent invention made by recombinant DNA techniques may result in theintroduction of N- or C-terminal residues encoded by linkers introducedto facilitate cloning or other manipulation steps. Other manipulationsteps include the introduction of linkers to join variable domains ofthe invention to further protein sequences including immunoglobulinheavy chains, other variable domains (for example, in the production ofdiabodies) or protein labels as discussed in more details below.

Although the embodiments illustrated in Examples comprise a “matching”pair of V_(H) and V_(L) domains, the invention also encompasses bindingfragments containing a single variable domain derived from either V_(H)or V_(L) domain sequences, especially V_(H) domains. In the case ofeither of the single chain specific binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain specific antigen-binding domain capable of binding GDF-8.This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed in WO92/01047 in which an individual colony containing either an H or L chainclone is used to infect a complete library of clones encoding the otherchain (L or H) and the resulting two-chain specific antigen-bindingdomain is selected in accordance with phage display techniques such asthose described in that reference. This technique is also disclosed inMarks et al., supra.

Antibodies can be conjugated by chemical methods with radionuclides,drugs, macromolecules, or other agents or can be made as fusion proteinscomprising one or more CDRs of the invention.

An antibody fusion protein contains a V_(H)-V_(L) pair where one ofthese chains (usually V_(H)) and another protein are synthesized as asingle polypeptide chain. These types of products differ from antibodiesin that they generally have an additional functional element; the activemoiety of a small molecule or the principal molecular structural featureof the conjugated or fused macromolecule.

In addition to the changes to the amino acid sequence outlined above,the antibodies can be glycosylated, pegylated, or linked to albumin or anonproteinaceous polymer. For instance, the presently disclosedantibodies may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodiesare chemically modified by covalent conjugation to a polymer to increasetheir circulating half-life, for example. Exemplary polymers, andmethods to attach them to peptides are also shown in U.S. Pat. Nos.4,766,106; 4,179,337; 4,495,285; and 4,609,546.

In other embodiments, the antibody may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original antibody. Addition ofglycosylation sites to the presently disclosed antibodies accomplishedby altering the amino acid sequence to contain glycosylation siteconsensus sequences are well known in the art. Another means ofincreasing the number of carbohydrate moieties on the antibodies is bychemical or enzymatic coupling of glycosides to the amino acid residuesof the antibody. These methods are described in WO 87/05330, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., 22: 259-306. Removalof any carbohydrate moieties present on the antibodies may beaccomplished chemically or enzymatically as described by Hakimuddin etal. (1987.) Arch. Biochem. Biophys., 259: 52; and Edge et al. (1981)Anal. Biochem., 118: 131 and by Thotakura et al. (1987) Meth. Enzymol.,138: 350.

Antibodies of the invention may also be tagged with a detectable orfunctional label. Detectable labels include radiolabels such as ¹³¹I or⁹⁹Tc, which may be attached to antibodies of the invention usingconventional chemistry known in the art. Labels also include enzymelabels such as horseradish peroxidase or alkaline phosphatase. Labelsfurther include chemical moieties such as biotin, which may be detectedvia binding to a specific cognate detectable moiety, e.g., labeledavidin.

Antibodies, in which CDR sequences differ only insubstantially fromthose set out in SEQ ID NO:n, wherein n is 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, or 48, are encompassed within the scope of thisinvention. Insubstantial differences include minor amino acid changes,such substitutions of 1 or 2 out of any 5 amino acids in the sequence ofa CDR. Typically, an amino acid is substituted by a related amino acidhaving similar charge, hydrophobic, or stereochemical characteristics.Such substitutions would be within the ordinary skills of an artisan.Unlike in CDRs, more substantial changes in structure framework regions(FRs) can be made without adversely affecting the binding properties ofan antibody. Changes to FRs include, but are not limited to, humanizinga non-human derived framework or engineering certain framework residuesthat are important for antigen contact or for stabilizing the bindingsite, e.g., changing the class or subclass of the constant region,changing specific amino acid residues which might alter an effectorfunction such as Fc receptor binding (Lund et al. (1991) J. Immun. 147:2657-2662 and Morgan et al. (1995) Immunology 86: 319-324), or changingthe species from which the constant region is derived. Antibodies mayhave mutations in the C_(H)2 region of the heavy chain that reduce oralter effector function, e.g., Fc receptor binding and complementactivation. For example, antibodies may have mutations such as thosedescribed in U.S. Pat. Nos. 5,624,821 and 5,648,260. In the IgG₁ or IgG₂heavy chain, for example, such mutations may be made at amino acidresidues 117 and 120 of SEQ ID NO:53, which represents the Fc portion ofIgG₁ (these residues correspond to amino acids 234 and 237 in thefull-length sequence of IgG₁ or IgG₂). Antibodies may also havemutations that stabilize the disulfide bond between the two heavy chainsof an immunoglobulin, such as mutations in the hinge region of IgG₄, asdisclosed in Angal et al. (1993) Mol. Immunol. 30:105-108.

D. Nucleic Acids, Cloning and Expression Systems

The present invention further provides an isolated nucleic acid encodingantibodies or binding fragments of the present invention. Nucleic acidaccording to the present invention may comprise DNA or RNA and may bewholly or partially synthetic. Reference to a nucleotide sequence as setout herein encompasses a DNA molecule with the specified sequence, andencompasses a RNA molecule with the specified sequence in which U issubstituted for T, unless context requires otherwise.

The nucleic acid of the invention comprises a coding sequence for a CDRor V_(H) or V_(L) domain of the invention as set forth herein.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone nucleic acid of the invention as above.

The present invention also provides a host cell, which comprises one ormore constructs as above. A nucleic acid encoding any CDR (H1, H2, H3,L1, L2, or L3), V_(H) or V_(L) domain, or specific antigen-bindingfragment as provided herein forms an aspect of the present invention, asdoes a method of production of the encoded product. The method comprisesexpression from the encoding nucleic acid. Expression may be achieved byculturing under appropriate conditions recombinant host cells containingthe nucleic acid. Following production by expression a V_(H) or V_(L)domain, or specific antigen-binding fragment may be isolated and/orpurified using any suitable technique, then used as appropriate.

Specific antigen-binding fragments, V_(H) and/or V_(L) domains, andencoding nucleic acid molecules and vectors according to the presentinvention may be provided isolated and/or purified, e.g., from theirnatural environment, in substantially pure or homogeneous form, or, inthe case of nucleic acid, free or substantially free of nucleic acid orgenes of origin other than the sequence encoding a polypeptide with therequired function.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, and yeast and baculovirus systems. Mammaliancell lines available in the art for expression of a heterologouspolypeptide include Chinese hamster ovary cells, HeLa cells, babyhamster kidney cells, NS0 mouse melanoma cells and many others. A commonbacterial host is E. coli. For cells suitable for producing antibodies,see Gene Expression Systems, eds. Fernandez et al., Academic Press,1999. Any cell compatible with the present invention may be used toproduce the presently disclosed antibodies.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids or viral,e.g., phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd ed., Sambrook etal., Cold Spring Harbor Laboratory Press, 1989. Many known techniquesand protocols for manipulation of nucleic acid, for example, inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, 2nd ed., Ausubel et al. eds., John Wiley & Sons, 1992.

Thus, a further aspect of the present invention provides a host cellcomprising nucleic acid as disclosed herein. A still further aspectprovides a method comprising introducing such nucleic acid into a hostcell. The introduction may employ any available technique. Foreukaryotic cells, suitable techniques may include calcium phosphatetransfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.,vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g., by culturing host cells under conditions forexpression of the gene.

E. Biological Deposits

E. coli cultures individually transformed with the phagemid vectorpCANTAB6 encoding nongermlined scFv's Myo29, Myo28, or Myo22 weredeposited on Oct. 2, 2002, at American Tissue Culture Collection (ATCC)under respective Deposit Designation Numbers PTA-4741, PTA-4740, andPTA-4739. The address of the depository is 10801 University Blvd,Manassas, Va. 20110, U.S.A.

II. Methods of Treating Disease and Other Uses

The antibodies of the present invention are useful to prevent, diagnose,or treat various medical disorders in humans or animals. The antibodiescan be used to inhibit or reduce one or more activities associated withGDF-8, or a related protein. Most preferably, the antibodies inhibit orreduce one or more of the activities of GDF-8 relative to the GDF-8 thatis not bound by an antibody. In certain embodiments, the activity ofGDF-8, when bound by one or more of the presently disclosed antibodies,is inhibited at least 50%, preferably at least 60, 62, 64, 66, 68, 70,72, 72, 76, 78, 80, 82, 84, 86, or 88%, more preferably at least 90, 91,92, 93, or 94%, and even more preferably at least 95% to 100% relativeto a mature GDF-8 protein that is not bound by one or more of thepresently disclosed antibodies. Inhibition of GDF-8 activity can bemeasured in pGL3(CAGA)₁₂ reporter gene assays (RGA) as described inThies et al. (Growth Factors (2001) 18: 251-259) and as illustrated inExamples 2 and 9, or in ActRIIB receptor assays as illustrated inExamples 3 and 6.

The medical disorder being diagnosed, treated, or prevented by thepresently disclosed antibodies is a muscle or neuromuscular disorder; anadipose tissue disorder such as obesity; type 2 diabetes; impairedglucose tolerance; metabolic syndromes (e.g., syndrome X); insulinresistance induced by trauma such as burns or nitrogen imbalance; orbone degenerative disease such as osteoporosis.

Other medical disorders being diagnosed, treated, or prevented by thepresently disclosed antibodies are disorders associated with a loss ofbone, which include osteoporosis, especially in the elderly and/orpostmenopausal women, glucocorticoid-induced osteoporosis, osteopenia,osteoarthritis, and osteoporosis-related fractures. Other targetmetabolic bone diseases and disorders include low bone mass due tochronic glucocorticoid therapy, premature gonadal failure, androgensuppression, vitamin D deficiency, secondary hyperparathyroidism,nutritional deficiencies, and anorexia nervosa. The antibodies arepreferably used to prevent, diagnose, or treat such medical disorders inmammals, especially, in humans.

The antibodies or antibody compositions of the present invention areadministered in therapeutically effective amounts. Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the severity of the medical condition inthe subject. The dosage may be determined by a physician and adjusted,as necessary, to suit observed effects of the treatment. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Antibodies that exhibit large therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any antibody usedin the present invention, the therapeutically effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test antibodywhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Levels in plasma may be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay. Examples of suitable bioassaysinclude DNA replication assays, transcription-based assays, GDF-8protein/receptor binding assays, creatine kinase assays, assays based onthe differentiation of pre-adipocytes, assays based on glucose uptake inadipocytes, and immunological assays.

Generally, the compositions are administered so that antibodies or theirbinding fragments are given at a dose from 1 μg/kg to 150 mg/kg, 1 μg/kgto 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg,100 μg to 1 mg/kg, and 500 μg/kg to 1 mg/kg. Preferably, the antibodiesare given as a bolus dose, to maximize the circulating levels ofantibodies for the greatest length of time after the dose. Continuousinfusion may also be used after the bolus dose.

The methods of treating, diagnosing, or preventing the above medicalconditions with the presently disclosed antibodies can also be used onother proteins in the TGF-β superfamily. Many of these proteins arerelated in structure to GDF-8, such as BMP-11. Accordingly, anotherembodiment provides methods of treating the aforementioned disorders byadministering to a subject an antibody capable of inhibiting BMP-11 oractivin, either alone or in combination with other TGF-β inhibitors,such as a neutralizing antibody against GDF-8. The antibodies of theinvention may also be used to treat a disease or condition associatedwith or mediated by BMP-11. See, e.g., U.S. Pat. Nos. 5,639,638 and6,437,111.

The antibodies of the present invention may be used to detect thepresence of proteins belonging to the TGF-β superfamily, such as BMP-11and GDF-8, in vivo or in vitro. By correlating the presence or level ofthese proteins with a medical condition, one of skill in the art candiagnose the associated medical condition. The medical conditions thatmay be diagnosed by the presently disclosed antibodies are set forthabove.

Such detection methods are well known in the art and, include ELISA,radioimmunoassay, immunoblot, Western blot, immunofluorescence,immunoprecipitation, and other comparable techniques. The antibodies mayfurther be provided in a diagnostic kit that incorporates one or more ofthese techniques to detect a protein (e.g., GDF-8). Such a kit maycontain other components, packaging, instructions, or other material toaid the detection of the protein and use of the kit.

Where the antibodies are intended for diagnostic purposes, it may bedesirable to modify them, for example, with a ligand group (such asbiotin) or a detectable marker group (such as a fluorescent group, aradioisotope or an enzyme). If desired, the antibodies (whetherpolyclonal or monoclonal) may be labeled using conventional techniques.Suitable labels include fluorophores, chromophores, radioactive atoms,electron-dense reagents, enzymes, and ligands having specific bindingpartners. Enzymes are typically detected by their activity. For example,horseradish peroxidase can be detected by its ability to converttetramethylbenzidine (TMB) to a blue pigment, quantifiable with aspectrophotometer. Other suitable labels may include biotin and avidinor streptavidin, IgG and protein A, and the numerous receptor-ligandcouples known in the art. Other permutations and possibilities will bereadily apparent to those of ordinary skill in the art, and areconsidered as equivalents within the scope of the instant invention.

Yet another aspect of the invention provides a method of identifyingtherapeutic agents useful in treatment of muscle and bone disorders.Appropriate screening assays, e.g., ELISA-based assays, are known in theart. In such a screening assay, a first binding mixture is formed bycombining an antibody of the invention and a ligand, e.g., GDF-8,BMP-11, activin; and the amount of binding between the ligand and theantibody in the first binding mixture (M₀) is measured. A second bindingmixture is also formed by combining the antibody, the ligand, and acompound or agent to be screened, and the amount of binding between theligand and the antibody in the second binding mixture (M₁) is measured.The amounts of binding in the first and second binding mixtures are thencompared, for example, by calculating the M₁/M₀ ratio. The compound oragent is considered to be capable of inhibiting GDF-8 activity if adecrease in binding in the second binding mixture as compared to thefirst binding mixture is observed. The formulation and optimization ofbinding mixtures is within the level of skill in the art, such bindingmixtures may also contain buffers and salts necessary to enhance or tooptimize binding, and additional control assays may be included in thescreening assay of the invention.

Compounds found to reduce the antibody-ligand binding by at least about10% (i.e., M₁/M₀<0.9), preferably greater than about 30%, may thus beidentified and then, if desired, secondarily screened for the capacityto inhibit GDF-8 activity in other assays such as the ActRIIB bindingassay (Example 2), and other cell-based and in vivo assays as describedin Examples 13, 15, and 16.

III. Pharmaceutical Compositions and Methods of Administration

The present invention provides compositions comprising the presentlydisclosed antibodies. Such compositions may be suitable forpharmaceutical use and administration to patients. The compositionstypically comprise one or more antibodies of the present invention and apharmaceutically acceptable excipient. As used herein, the phrase“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.The compositions may also contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions. Thepharmaceutical compositions may also be included in a container, pack,or dispenser together with instructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes. The administration may, forexample, be intravenous, intraperitoneal, intramuscular, intracavity,subcutaneous or transdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor™ EL (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, and sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theantibodies can be incorporated with excipients and used in the form oftablets, or capsules. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, and the like can contain any of the followingingredients, or compounds of a similar nature; a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel™, or corn starch; a lubricant such as magnesium stearate orSterotes™; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, antibodies are delivered in the formof an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For example, in case of antibodies that comprise the Fc portion,compositions may be capable of transmission across mucous membranes(e.g., intestine, mouth, or lungs) via the FcRn receptor-mediatedpathway (U.S. Pat. No. 6,030,613). Transmucosal administration can beaccomplished, for example, through the use of lozenges, nasal sprays,inhalers, or suppositories. For transdermal administration, the activecompounds are formulated into ointments, salves, gels, or creams asgenerally known in the art. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, detergents, bile salts, and fusidic acidderivatives.

The presently disclosed antibodies may be prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Liposomal suspensions containing the presentlydisclosed antibodies can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It may be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art offormulating such an active compound for the treatment of individuals.

The following examples provide illustrative embodiments of the inventionwhich do not in any way limit the invention. One of ordinary skill inthe art will recognize the numerous other embodiments are encompassedwithin the scope of the invention.

The entire contents of all references, patents and published patentapplications cited throughout this application are herein incorporatedby reference.

EXAMPLES Example 1 Purification of GDF-8

Conditioned media from a selected cell line expressing recombinant humanGDF-8 protein (mature GDF-8 and GDF-8 propeptide) was acidified to pH6.5 and applied to a 80×50 mm POROS™ HQ anion exchange column in tandemto a 80×50 mm POROS™ SP cation exchange column (PerSeptive Biosystems,Foster City, Calif.). The flow through was adjusted to pH 5.0 andapplied to a 75×20 mm POROS™ SP cation exchange column (PerSeptiveBiosystems) and eluted with a NaCl gradient. Fractions containing theGDF-8 latent complex, as confirmed by SDS-PAGE, were pooled, acidifiedwith trifluoroacetic acid (TFA) to pH 2-3, then brought up to 200 mlwith 0.1% TFA to lower the viscosity. The pool was then applied to a250×21.2 mm C₅ column (Phenomenex, Torrance, Calif.) preceded by a60×21.2 mm guard column (Phenomenex) and eluted with a TFA/acetonitrilegradient, to separate mature GDF-8 from GDF-8 propeptide. Pooledfractions containing mature GDF-8 were concentrated by lyophilization toremove the acetonitrile and 20 ml of 0.1% TFA was added. The sample wasthen applied to a 250×10 mm C₅ column (Phenomenex) heated to 60° C. toaid in separation. This was repeated until further separation could nolonger be achieved. Fractions containing mature GDF-8 were then pooledand brought up to 40% acetonitrile and applied to a 600×21.2 BioSep™S-3000 size exclusion column (Phenomenex) preceded by a 60×21.2 guardcolumn. Fractions containing purified mature GDF-8 were pooled andconcentrated for use in subsequent experiments.

On SDS-PAGE, purified mature GDF-8 migrated as a broad band at 25 kDaunder nonreducing conditions and 13 kDa under reducing conditions. Asimilar SDS-PAGE profile has been reported for murine GDF-8 by McPherronet al. (Proc. Nat. Acad. Sci. U.S.A. (1997) 94: 12457-12461) andreflects the dimeric nature of the mature protein. The active matureBMP-11 dimer was purified from conditioned media from a cell lineexpressing recombinant human BMP-11 in a similar manner.

Active mature BMP-11 was purified from conditioned media from a cellline expressing recombinant human GDF-8 propeptide/mature BMP-11chimeric protein. The conditioned medium was loaded onto a 10 ml TALON™column (Clonetech, Palo Alto, Calif.) in 50 mM Tris pH 8.0, 1 M NaCl at1 ml/min. The bound protein was eluted with a 50 mM Tris pH 8.0, 1 MNaCl, 500 mM Imidazole. Pooled fractions containing the GDF-8propeptide/BMP-11 latent complex were acidified with 10% TFA to a pH of3. The pool was then applied to a 250×4.6 mm Jupiter C4 column(Phenomenex, Torrance, Calif.) which was heated to 60° C. for betterseparation of mature BMP-11 and GDF-8 propeptide, and eluted with aTFA/acetonitrile gradient. Pooled fractions containing mature BMP-11were concentrated by lyophilization. On SDS-PAGE, purified mature BMP-11migrated at 25 kDa under non-reducing conditions and at 12 kDa underreducing conditions.

Example 2 Biological Activity of Purified Recombinant Human GDF-8

To demonstrate the activity of GDF-8, a reporter gene assay (RGA) wasdeveloped using a reporter vector pGL3(CAGA)₁₂ expressing luciferase.The CAGA sequence was previously reported to be a TGF-β responsivesequence within the promoter of the TGF-β induced gene PAI-1 (Denner etal. (1998) EMBO J., 17: 3091-3100).

A reporter vector containing 12 CAGA boxes was made using the basicluciferase reporter plasmid pGL3 (Promega, Madison, Wis.). The TATA boxand transcription initiation site from the adenovirus major laterpromoter (−35/+10) was inserted between the BgIII and HindIII sites.Oligonucleotides containing 12 repeats of the CAGA boxes AGCCAGACA wereannealed and cloned into the XhoI site. The human rhabdomyosarcoma cellline A204 (ATCC HTB-82) was transiently transfected with pGL3(CAGA)₁₂using FuGENE™ 6 transfection reagent (Boehringer Manheim, Germany).Following transfection, cells were cultured on 48 well plates in McCoy's5A medium supplemented with 2 mM glutamine, 100 U/ml streptomycin, 100μg/ml penicillin and 10% fetal calf serum for 16 hrs. Cells were thentreated with or without 10 ng/ml GDF-8 in McCoy's 5A media withglutamine, streptomycin, penicillin, and 1 mg/ml bovine serum albuminfor 6 hrs at 37° C. Luciferase was quantified in the treated cells usingthe Luciferase Assay System (Promega).

FIG. 4A shows that GDF-8 maximally activated the reporter construct10-fold, with an ED50 of 10 ng/ml, indicating that purified recombinantGDF-8 was biologically active. BMP-11 and activin elicited a similarbiological response.

Example 3 Binding Properties of Purified. GDF-8 in the ActRIIB BindingAssay

The GDF-8 latent complex was biotinylated at a ratio of 20 moles ofEZ-link Sulfo-NHS-Biotin (Pierce, Rockford, Ill., Cat. No. 21217) to 1mole of the GDF-8 complex for 2 hours on ice. The reaction wasterminated by dropping the pH using 0.5% TFA and the complex wassubjected to chromatography on a C₄ Jupiter 250×4.6 mm column(Phenomenex) to separate mature GDF-8 from GDF-8 propeptide.Biotinylated mature GDF-8 fractions eluted with a TFA/CH₃CN gradientwere pooled, concentrated and quantified by MicroBCA™ protein AssayReagent Kit (Pierce, Rockford, Ill., Cat. No. 23235).

Biotinylated mature BMP-11 was prepared from BMP-11 latent complex inthe same manner as described above. Recombinant ActRIIB-Fc chimera (R&DSystems, Minneapolis, Minn., Cat. No. 339-RB/CF) was coated on 96-wellflat-bottom assay plates (Costar, N.Y., Cat. No. 3590) at 1 μg/ml in 0.2M sodium carbonate buffer overnight at 4° C. Plates were then blockedwith 1 mg/ml bovine serum albumin and washed following standard ELISAprotocol. 100 μl aliquots of biotinylated GDF-8 or BMP-11 at variousconcentrations were added to the blocked ELISA plate, incubated for 1hr, washed, and the amount of bound GDF-8 or BMP-11 was detected byStreptavidin-Horseradish peroxidase (SA-HRP, BD PharMingen, San Diego,Calif., Cat. No. 13047E) followed by the addition of TMB (KPL,Gaithersburg, Md., Cat. No. 50-76-04). Colorimetric measurements weredone at 450 nM in a Molecular Devices microplate reader.

As shown in FIG. 1, biotinylated GDF-8 and BMP-11 bound to ActRIIB, theputative GDF-8 type II receptor with an ED₅₀ of 15 and 40 ng/ml;respectively, indicating that the ActRIIB binding assay is a sensitivein vitro binding assay for GDF-8 and BMP11.

Example 4 Isolation of Myo22 by Panning of scFv Libraries on GDF-8

An scFv phagemid library, which is an expanded version of the 1.38×10¹⁰library described (Vaughan et al. (1996) Nature Biotech., 14: 309-314),was used to select antibodies specific for GDF-8. Soluble GDF-8 protein(at 10 μg/ml in 50 mM sodium carbonate buffer, pH 9.6) was coated ontowells of a microtitre plate overnight at 4° C. Wells were washed in PBSand blocked for 2 hrs at 37° C. in MPBS (3% Marvel™ skimmed milk powderin PBS). Purified phage (10¹² transducing units (tu)) in 100 l of 3%MPBS were added to blocked wells and incubated at room temperature for 1hour. Wells were washed 10 times with PBST (PBS containing 0.1% v/vTween™ 20), then 10 times with PBS. Bound phage particles were elutedwith 100 μl of 100 mM triethylamine for 10 minutes at room temperature,then immediately neutralized with 50 μl of 1 M Tris-HCl pH 7.4. Theeluted phage was used to infect 10 ml exponentially growing E. coli TG1.Infected cells were grown in 2TY broth for 30 minutes at 37° C.stationary, followed by 30 minutes at 37° C. with aeration, thenstreaked onto 2TYAG plates and incubated overnight at 30° C. Colonieswere scraped off the plates into 10 ml 2TY broth and 15% glycerol addedfor storage at −70° C.

Glycerol stock cultures from the first round panning selection weresuperinfected with helper phage and rescued to give scFvantibody-expressing phage particles for the second round of panning. Atotal of three rounds of panning were carried out in this way.

Example 5 Selection of Myo28 and Myo29 from scFv Libraries

Soluble selections were carried out using biotinylated GDF-8 protein(bioGDF-8). BioGDF-8 was used at a concentration of 1 μg/ml. An scFvlibrary, as described in Example 4, was used. Purified scFv phage (10¹²tu) in 100 μl 3% MPBS were blocked for 30 minutes, then biotinylatedantigen was added and incubated at room temperature for 1 hour.Phage/antigen was added to 50 μl of Dynal™ M280 streptavidin magneticbeads that had been blocked for 1 hour at 37° C. in 1 ml of 3% MPBS andincubated for a further 15 minutes at room temperature. Beads werecaptured using a magnetic rack and washed four times in 1 ml of 3% MPBSwith 0.1% (v/v) Tween™ 20 followed by three washes in PBS. After thelast PBS wash, beads were resuspended in 100 μl PBS and used to infect 5ml exponentially growing E. coli TG-1 cells. Cells and phage wereincubated for 1 hour at 37° C. (30 minutes stationary, 30 minutesshaking at 250 rpm), and then spread on 2TYAG plates. Plates wereincubated at 30° C. overnight and colonies visualized the next day.Output colonies were scraped off the plates and phage rescued asdescribed above. A second round of soluble selection was carried out asdescribed above.

Example 6 ActRIIB Receptor Inhibition Assay and Screen

Output colonies, obtained as described in Examples 4 and 5, were pickedinto 96 well plates containing 100 μl of 2TYAG. ScFv production wasinduced by addition of 1 mM IPTG to exponentially growing cultures andincubation overnight at 30° C. Crude scFv-containing culturesupernatants were screened for the ability to inhibit the binding ofbioGDF-8 to ActRIIB essentially as described in Example 3. The assay wasmodified slightly in that binding of bioGDF-8 was detected withEuropium-labeled streptavidin and using the DELFIA™ reagent kit(PerkinElmer Life Sciences, Boston, Mass.) in time-resolved fluorometricassays (TRF). Positive clones, showing inhibition of binding signalgreater than irrelevant clones, were picked and assayed to confirmactivity.

Purified scFv from positive clones identified from the receptorinhibition screen was tested in the inhibition assay as above. Atitration of scFv concentrations was used in order to establish clonepotency as measured by IC₅₀ values in the assay. The results of theexperiments are shown in FIG. 2. As determined in these assays, IC₅₀ forscFv's of Myo29, Myo28, and Myo22 are 2.4 nM, 1.7 nM, and 60 nM,respectively. Therefore, these antibodies are potent inhibitors of GDF-8activity.

Example 7 Specificity Characterization by Phage ELISA

To determine the specificity of antibodies, a phage ELISA was performedfor positive clones from the ActRIIB screen against GDF-8 and unrelatedproteins. Individual E. coli colonies containing phagemid wereinoculated into 96 well plates containing 100 μl 2TYAG medium per well.M13K07 helper phage was added to a multiplicity of infection (moi) of 10to exponentially growing culture and the plates incubated a further 1hour at 37° C. Plates were centrifuged in a benchtop centrifuge at 2000rpm for 10 minutes. The supernatant was removed and cell pellets wereresuspended in 100 μl 2TYAK and incubated at 30° C. overnight withshaking. The next day, plates were centrifuged at 2000 rpm for 10minutes and 100 μl phage-containing supernatant from each welltransferred to a fresh 96 well plate. Phage samples were blocked in afinal concentration of 3% MPBS for 1 hour at room temperature, prior toELISA.

GDF-8 or irrelevant protein was coated overnight at 4° C. onto 96-wellmicrotiter plates at 1 μg/ml. After coating, the solutions were removedfrom the wells, and the plates blocked for 1 hour at room temperature in3% MPBS. Plates were rinsed with PBS then 50 μl of pre-blocked phageadded to each well. The plates were incubated at room temperature for 1hour and then washed with 3 changes of PBST followed by 3 changes ofPBS. To each well, 50 μl of a 1:5000 dilution of anti-M13-HRP conjugate(Pharmacia) was added and the plates incubated at room temperature for 1hour. Each plate was washed three times with PBST then 3 times with PBS.Fifty microliters of TMB substrate was added to each well and incubateduntil color development. The reaction was stopped by the addition of 25μl of 0.5 M H₂SO₄. The signal generated was measured by reading theabsorbance at 450 nm using a microtiter plate reader. Specific bindingto GDF-8 was confirmed.

Example 8 Sequencing of scFv, Conversion to IgG, and Germlining

Neutralizing scFv E. coli clones were streaked out onto 2TYAG plates andincubated overnight at 30° C. Triplicate colonies from these plates weresequenced using pCANTAB6 vector sequence oligos to amplify the V_(H) andV_(L) regions from the scFv clone. DNA sequences of the scFv fragmentsused for making Myo29, Myo28, and Myo22 IgG's are represented by SEQ IDNO:13, SEQ ID NO:7, and SEQ ID NO:1, respectively.

Heavy and light chain V regions from scFv clones were amplified usingPCR and clone-specific primers. PCR products were digested withappropriate restriction enzymes and subcloned into vectors containinghuman IgG₁ heavy chain constant domain (for V_(H) domains) or vectorscontaining human lambda light chain constant domain as appropriate (forV_(L) domains). Correct insertion of V region domains into plasmids wasverified by sequencing of plasmid DNA from individual E. coli colonies.Plasmids were prepared from E. coli cultures by standard techniques andheavy and light chain constructs co-transfected into COS cells usingstandard techniques. Secreted IgG was purified using Protein A Sepharose(Pharmacia, Peapack, N.J.) and buffer exchanged into PBS.

Sequence data for the scFv clones was used to identify the nearestgermline sequence for the heavy and light chain of each clone.Appropriate mutations were made using standard site directed mutagenesistechniques with the appropriate mutagenic primers. Mutation of scFvsequences was confirmed by sequence analysis. Germlined scFv and V_(H)and V_(L) domain sequences for Myo28 and Myo29 are set forth in SEQ IDNO:19 and SEQ ID NO:25, respectively.

Example 9 Biological Activity of Antibodies

FIG. 3A shows that preincubation of Myo29 with biotinylated GDF-8 at 10ng/ml inhibited GDF-8 binding to ActRIIB in the ActRIIB binding assay,as described in Example 3, with an IC₅₀ of 0.2-0.4 nM. Similarly in FIG.3B, Myo29 inhibited biotinylated BMP-11 binding to ActRIIB with the sameIC₅₀.

Myo29 also blocked GDF-8 activity in an in vitro bioassay. By way ofexample, when GDF-8 was preincubated with Myo29 for 1 hour at roomtemperature, the biological activity of GDF-8 was reduced as determinedin RGA assays performed essentially as described in Example 2. FIG. 4Cshows induction of pGL3(CAGA)₁₂ reporter activity at the ED₅₀ for GDF-8,20 ng/ml, in the presence of Myo29. Myo29 reduced the GDF-8 induction ina dose-responsive manner, with an IC₅₀ of 15-30 ng/ml (0.1-0.2 nM).Myo29 also inhibited the biological activity of BMP-11 to the sameextent (FIG. 4B). In contrast, the activity of activin in this assay wasnot affected by Myo29 (FIG. 4D), presumably due to the relatively lowhomology between GDF-8 and activin, as compared to GDF-8 and BMP-11.

Myo22 and Myo28 were also tested in the RGA and ActRIIB binding assays.Both antibodies block GDF-8 and BMP-11 activity. The IC₅₀ for Myo28, forexample, is 0.2-0.35 nM.

Example 10 Mapping of Epitopes for Myo22, Myo28, and Myo29

In order to map the exact epitope of the antibodies, 48 overlapping13-residue peptides representing the entire sequence of mature GDF-8 setforth in SEQ ID NO:49 were synthesized directly on cellulose paper usingthe spot synthesis technique (Molina et al. (1996) Peptide Research,9:151-155; Frank et al. (1992) Tetrahedron, 48: 9217-9232). The overlapof the peptides was 11 amino acids. In this array, cysteine residueswere replaced with serine in order to reduce the chemical complicationsthat are caused by the presence of cysteines. Cellulose membranesmodified with polyethylene glycol and Fmoc-protected amino acids werepurchased from Abimed (Lagenfeld, Germany). The array was defined on themembrane by coupling a β-alanine spacer and peptides were synthesizedusing standard DIC (diisopropylcarbodiimide)/HOBt (hydroxybenzotriazole)coupling chemistry as described previously (Molina et al. (1996) PeptideResearch, 9: 151-155; Frank et al. (1992) Tetrahedron, 48: 9217-9232).

Activated amino acids were spotted using an Abimed ASP 222 robot.Washing and deprotection steps were done manually and the peptides wereN-terminally acetylated after the final synthesis cycle. Followingpeptide synthesis, the membrane was washed in methanol for 10 minutesand in blocker (TBST (Tris-buffered saline with 0.1% (v/v) Tween™ 20)and 1% (w/v) casein) for 10 minutes. The membrane was then incubatedwith 2.5 μg/ml of an anti-GDF-8 antibody in blocker for 1 hour withgentle shaking. After washing with blocker 3 times for 10 minutes, themembrane was incubated with HRP-labeled secondary antibody (0.25 μg/mlin blocker) for 30 minutes. The membrane was then washed three times for10 minutes each with blocker and 2 times for 10 minutes each with TBST.Bound antibody was visualized using SuperSignal™ West reagent (Pierce)and a digital camera (Alphananotech Fluoromager). Results are shown inFIG. 5. In particular, as seen from FIG. 5, the epitope for Myo29 wasmapped between amino acids 72 and 88 of mature GDF-8. Myo22, on theother hand, recognizes an epitope within the first 44 N-terminal aminoacids in the sequence of mature GDF-8 (amino acids 1 through 44 of SEQID NO:49). Finally, the epitope for Myo28 comprises residues locatedwithin the first 98 N-terminal amino acids of mature GDF-8.

In order to further characterize the Myo29 epitope, deletion andsubstitution analyses were performed using spot synthesis. In thesubstitution analysis, each residue of this peptide was individuallyreplaced with each of the 20 natural amino acids except cysteine.Synthesis and binding assays were performed as described above. Theresults are shown in FIG. 6, wherein the first row, first two columnsand last three columns represent wild-type peptide controls. The resultsdemonstrate that when Lys-78, Pro-81, and Asn-83 are each individuallymutated to another amino acid, the binding affinity of Myo29 to thepeptide is significantly reduced. Therefore, Myo29 recognizes a sequencecomprising Lys-Xaa1-Xaa2-Pro-Xaa3-Asn (SEQ ID NO:54), wherein Xaa1,Xaa2, and Xaa3 each is either any amino acid, or Xaa1=Met, Xaa2=Ser, andXaa3=Ile, independently of each other.

Example 11 Immunoprecipitation of GDF-8

In order to evaluate the binding of Myo29 and Myo28 to mature GDF-8 andGDF-8 complexes, an immunoprecipitation study was conducted. CHO cellsexpressing GDF-8 were labeled with ³⁵S-methionine and ³⁵S-cysteine. 100μl conditioned media from these cells, containing GDF-8 protein (matureGDF-8 and latent complex) was incubated with 20 μg/ml Myo29 or Myo28 for1 hour at 4° C. Protein A-Sepharose™ was added and incubated overnightat 4° C. The immunoprecipitate was collected, resuspended in reducingsample buffer and analyzed by SDS-PAGE. The gel was fixed, enhanced withautoradiography enhancer solution, dried, and the autorad was developed.FIG. 7 shows that both Myo29 and Myo28 can immunoprecipitate matureGDF-8, the GDF-8 latent complex and unprocessed GDF-8. Both antibodiesbind to GDF-8 dimer under non-reducing conditions as determined byWestern blotting.

Example 12 Pharmacokinetics

The pharmacokinetics (PK) of Myo29 was evaluated in C57B6/SCID mice at adose of 1 mg/kg after a single intravenous (IV) or intraperitoneal (IP)administration. The animals received a mixture of unlabeled and¹²⁵I-labeled Myo29 at the dose listed above and serum concentrationswere determined based on ¹²⁵I radioactivity in the serum and thespecific activity of the injected dose. FIG. 8 shows a plot of serumconcentration versus time for Myo29 administered either IV or IP.

Myo29 showed a prolonged terminal half-life of around one week and lowclearance around 1 ml/hr/kg. Initial volume of distribution was about 83ml/kg. Apparent volume of distribution was about 227 ml/kg. Myo29reached a peak concentration at about 6 hrs post injection. Fractionabsorbed following IP injection was about 77%.

Example 13 In Vivo Effect of Myo29 on Muscle Mass and Strength

In order to determine whether Myo29 blocks GDF-8 activity in vivo, Myo29was tested in adult SCID mice. SCID mice suffer from a severe combinedimmune deficiency, and therefore do not generate an immunologicalreaction following injections of human antibodies such as Myo29. Musclemass was used as an indicator for GDF-8 activity in mice treated withMyo29.

Male C57B6 SCID eight weeks old mice were weighed and evenly distributedwith respect to body weight into groups of eight. Myo29 in PBS bufferwas injected into the mice intraperitoneally at various doses (60, 10,and 1 mg/kg) weekly. A double dose was given the first week. Vehicle(PBS)-treated or untreated mice were used as controls. The treatmentscontinued for four weeks. Muscle mass was assessed by dissecting andweighing the gastrocnemius and quadriceps following treatment. Afterfour weeks of treatment, muscle mass was increased in all groups treatedwith Myo29, ranging from 10% to 23%, with groups treated with higherdoses reaching significant levels (FIG. 9, p<0.01).

In another experiment, female CB17 SCID mice were treated with Myo29weekly at various doses (10, 5, 2.5, and 1 mg/kg) for 4 or 12 weeks.Again, treatments with Myo29 for 4 weeks resulted in an increase ingastrocnemius and quadriceps weight ranging from 10% to 20% (FIGS. 10Aand 10B). Longer treatment (12 weeks) resulted in greater increases inmuscle mass (12% to 28%) with all groups treated with Myo29 reachingstatistically significant levels (FIGS. 11A and 11B).

In order to determine whether increased muscle mass leads to strongermuscles, muscle strength of front limb was measured with a grip strengthtest meter (model 1027 csx, Columbus Instruments, Columbus, Ohio). After12 weeks of treatment, the front limb strength was 17% and 23% higher inmice treated with 5 mg/kg or 10 mg/kg of Myo29 respectively as comparedto the vehicle control (p<0.01, FIG. 12). The results of this studydemonstrate that Myo29 inhibits GDF-8 activity in vivo resulting insignificant increases in muscle mass and muscle strength.

Example 14 Treatment of Metabolic Disorders

Inhibitors of GDF-8, such as, for example inhibitory antibodies, areuseful for treatment of metabolic disorders such as type 2 diabetes,impaired glucose tolerance, metabolic syndrome (e.g., syndrome X),insulin resistance induced by trauma (e.g., burns or nitrogenimbalance), and adipose tissue disorders (e.g., obesity). The anti-GDF-8antibodies of the invention are used to treat a subject at disease onsetor having an established metabolic disease.

Efficacy of anti-GDF-8 antibodies for treatment of metabolic disorders,e.g., type 2 diabetes and/or obesity, is confirmed using wellestablished murine models of obesity, insulin resistance and type 2diabetes, including ob/ob, db/db, and strains carrying the lethal yellowmutation. Insulin resistance can also be induced by high fat or highcaloric feeding of certain strains of mice, including C57BL/6J.Similarly to humans, these rodents develop insulin resistance,hyperinsuliemia, dyslipidemia, and deterioration of glucose homeostasisresulting in hyperglycemia. Outcome assessments are based on serummeasurements of glucose, insulin and lipids. Measures of improvedinsulin sensitivity can be determined by insulin tolerance tests andglucose tolerance tests. More sensitive techniques would include the useof euglycemic-hyperinsulinemic clamps for assessing improvements isglycemic control and insulin sensitivity. In addition, the clamptechniques would allow a quantitative assessment of the role of themajor glucose disposing tissues, (muscle, adipose, and liver), inimproved glycemic control.

In one study, treatment with an anti-GDF-8 antibody such as Myo29 (IPinjection) or vehicle is conducted for one week to six months. Thetreatment protocol could vary, with testing of different doses andtreatment regimens (e.g., daily, weekly, or bi-weekly injections). It isanticipated that mice treated with the anti-GDF-8 antibody would havegreater glucose uptake, increased glycolysis and glycogen synthesis,lower free fatty acids and triglycerides in the serum as compared tomice receiving placebo treatment.

The inhibitory antibodies against GDF-8 are also used to prevent and/orto reduce severity and/or the symptoms of the disease. It is anticipatedthat the anti-GDF-8 antibodies would be administered as a subcutaneousinjection as frequently as once per day and as infrequently as once permonth. Treatment duration could range from one month to several years.

To test the clinical efficacy of anti-GDF-8 in humans, subjectssuffering from or at risk for type 2 diabetes are identified andrandomized to a treatment group. Treatment groups include a placebogroup and one to three groups receiving antibody (different doses).Individuals are followed prospectively for one month to three years toassess changes in glucose metabolism. It is anticipated that individualsreceiving treatment would exhibit an improvement.

The antibodies are administered as the sole active compound or incombination with another compound or composition. When administered asthe sole active compound or in combination with another compound orcomposition, the dosage is preferably from approximately 1 μg/kg to 20mg/kg, depending on the severity of the symptoms and the progression ofthe disease. The appropriate effective dose is selected by a treatingclinician from the following ranges: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg,100 μg to 1 mg/kg, and 500 μg/kg to 1 mg/kg. Exemplary treatmentregimens and outcomes are summarized in Table 3.

TABLE 3 Examples of Clinical Cases Patient Status prior to Treatment No.treatment Regimen Outcome Patient No clinical signs, 0.01-1 mg/kg everyPrevention of type 2 1 family history of 4 weeks for 48 diabetes type 2diabetes weeks Patient Mild clinical 0.01-20 mg/kg Improved insulin 2signs of weekly for 4 more tolerance and syndrome X weeks glucosemetabolism, and lower blood pressure Patient Advanced stage 0.01-20mg/kg twice Improvement of 3 of type 2 weekly for 6 or more clinicalsigns, diabetes weeks reduction in severity of symptoms and/or increasein muscle mass/body fat ratio Patient Severe insulin 0.01-20 mg/kg dailyImprovement of 4 resistance for 6 or more weeks clinical signs,and/obesity reduction in severity of symptoms and/or decrease in bodyfat

The specification is most thoroughly understood in light of theteachings of the references cited within the specification, all of whichare hereby incorporated by reference in their entirety. The embodimentswithin the specification provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention. The skilled artisan recognizes that many other embodimentsare encompassed by the claimed invention and that it is intended thatthe specification and examples be considered as exemplary only, with atrue scope and spirit of the invention being indicated by the followingclaims.

1. An isolated antibody capable of specifically binding GDF 8 or BMP 11comprising: (a) the amino acid sequence of SEQ ID NO:14, (b) the aminoacid sequence of SEQ ID NO:26, (c) a fragment of the amino acid sequenceof SEQ ID NO: 14 that is capable of specifically binding GDF 8 or BMP11, or (d) a fragment of the amino acid sequence of SEQ ID NO:26 that iscapable of specifically binding GDF 8 or BMP
 11. 2. The antibody ofclaim 1, comprising the amino acid sequence of any one of SEQ ID NO:16,SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
 3. The antibody of claim1, wherein the antibody is capable of specifically binding to a proteincomprising the amino acid sequence set forth in SEQ ID NO:54.
 4. Theantibody of claim 3, wherein SEQ ID NO:54 is characterized by at leastone of the following: (a) the second amino acid of SEQ ID NO:54 ismethionine; (b) the third amino acid of SEQ ID NO:54 is serine; and (c)the fifth amino acid of SEQ ID NO:54 is isoleucine.
 5. The antibody ofclaim 1, wherein the antibody is human.
 6. The antibody of claim 1,wherein the antibody is IgG₁ or lgG₄.
 7. The antibody of claim 1,wherein the amino acid sequence of the antibody comprises SEQ ID NO: 53.8. The antibody of claim 7, wherein the amino acid sequence is modifiedat residues corresponding to amino acid 117 or amino acid 120 of SEQ IDNO:53.
 9. The antibody of claim 1, wherein the antibody is IgG_(1λ)orIgG_(1K).
 10. A pharmaceutical composition, comprising the antibody ofclaim
 1. 11. The antibody of claim 1, produced by the steps of: (a)providing a starting repertoire of nucleic acids encoding a variabledomain which either include a CDR3 to be replaced or lack a CDR3encoding region; (b) combining the repertoire with a donor nucleic acidencoding an amino acid sequence substantially as set out in any one ofSEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,and SEQ ID NO:36, such that the donor nucleic acid is inserted into theCDR3 region in the repertoire so as to provide a product repertoire ofnucleic acids encoding a variable domain; (c) expressing the nucleicacids of the product repertoire; (d) selecting a specific antigenbinding fragment specific for GDF 8; and (e) recovering the specificantigen binding fragment or nucleic acid encoding the binding fragment.12. The antibody of claim 1, wherein the antibody is capable ofinhibiting binding of GDF 8 to ActRIIB.
 13. The antibody of claim 1,wherein the fragment of SEQ ID NO:14 comprises amino acids 1 to 117 ofSEQ ID NO:14 (SEQ ID NO:16).
 14. The antibody of claim 1, wherein thefragment of SEQ ID NO:14 comprises amino acids 135 to 239 of SEQ IDNO:14 (SEQ ID NO:18).
 15. The antibody of claim 1, wherein the fragmentof SEQ ID NO:14 comprises amino acids 31 to 35 of SEQ ID NO:14 (SEQ IDNO:31).
 16. The antibody of claim 1, wherein the fragment of SEQ IDNO:14 comprises amino acids 50 to 66 of SEQ ID NO:14 (SEQ ID NO:32). 17.The antibody of claim 1, wherein the fragment of SEQ ID NO:14 comprisesamino acids 99 to 106 of SEQ ID NO:14 (SEQ ID NO:33).
 18. The antibodyof claim 1, wherein the fragment of SEQ ID NO:14 comprises amino acids157 to 167 of SEQ ID NO:14 (SEQ ID NO:34).
 19. The antibody of claim 1,wherein the fragment of SEQ ID NO:14 comprises amino acids 183 to 189 ofSEQ ID NO:14 (SEQ ID NO:35).
 20. The antibody of claim 1, wherein thefragment of SEQ ID NO:14 comprises amino acids 222 to 228 of SEQ IDNO:14 (SEQ ID NO:36).
 21. The antibody of claim 1, wherein the fragmentof SEQ ID NO:26 comprises amino acids 135 to 239 of SEQ ID NO:26 (SEQ IDNO:30).